We have undertaken an immunogenetic analysis of the signals required for the primary in vitro generation of MHC class II-specific murine CTL. By varying the genetic relationship between responding and stimulating cells in mixed leukocyte cultures, we have demonstrated that optimal generation of class II-specific CTL requires the absence of a simultaneous class I MHC stimulus in the cultures. The inhibitory effect of a class I-specific response on the generation of class II-specific CTL in these studies: (i) is not due to preferential consumption of helper T cell-derived lymphokines by class I-specific cells; (ii) is not due to elimination of class II antigen-bearing stimulator cells by class I-specific cells; (iii) is not mediated via signals requiring linked recognition of class I and class II alloantigens on stimulator cells. The inhibitory cell has been identified by depletion experiments as an Lyt2+ cell in the responder spleen population. The action of this Lyt2+ inhibitory cell can be blocked during culture by monoclonal alpha Lyt2 antibody in the absence of complement, which does not block the generation of class I-specific CTL. These results suggest that the class I-specific Lyt2+ inhibitory cell detected in this system either is not a CTL, or is an Lyt2-independent CTL.